Method of producing 1,4-diene-3-ketosteroids



United States Patent-O see, Germany, assiguors to Schering A.G., Berlin, Germany No Drawing. Filed May 2, 1961, Ser. No. 107,029

Claims priority, application Germany May 20, 1960 27 Claims. (Cl. 195-51) The present invention relates to a method of producing 1,4-diene-3-ketosteroids, and more particularly to a new method Which results in the production of such 1,4- diene-3-ketosteroids in high yield and also starting from compounds which could not previously be used as starting materials for the production of such final products, as Well as to the production of new compounds.

The various perhydrocyclopcntenophenanthrenes which simultaneously have double bonds in the 1-position and in the 4-position, for example prednisolone, prednisone, triamcmolone, A -androstadiene-3,l7-dione, (and many others, possess valuable properties which make these compounds of great interest as therapeutic products and also as intermediate products for the production of theraeutic steroid products.

Methods of producing these compounds comprise not only chemical methods but also microbiological methods, the latter having the advantage also that they are applicable to compounds which are sensitive against chemical reagents, and in addition often gives rise to considerably improved yields. Not only bacteria but also fungi have been used for the introduction of a A -double bond.

The species belonging to the family Bacillaceae have the common property of spore formation. With respect to the biochemical activity not only do the various species difier from each other, but also the strains of each species vary considerably, and in contrast to the fungi it is not possible by placing bacteria in its place in the system to predict what its biochemical activity would be since often the origin of the individual strain of a species is of considenable importance With respect to its bio chemical activity.

Thus, for example, a great number of hithertoavailable strains of Bacillus subtilis (Naturwissenschaften, 43, page 39) do not possess the property which they would be expected to possess according to the publication, namely of dehydrogenating a steroid hormone in 1,2- position.

In the case of the species Bacillus sphaericus, which has been used for a long time for the purpose of 1,2-dehydrogenation in the steroid series, it has been shown, that besides very slow reaction speed, that the success of the A -introduction is greatly dependent on the structure of the used starting material. Thus, for examplegonly A -3-ketosteroids can be converted to the corresponding A -compound, but A -3-hydroxy steroids cannot be so converted.

It is also known that microorganisms of the family Corynebacteriaceae, the species Corynebacterium mediolanum and Corynebacterium simplex are useful for 1,2- de'hydrogenation and also .for the conversion of dehydroepiandrosterone into A 'androstadiene 3,17 dione. However, with these microorganisms only extremely moderate yields can be obtained and these require a long time because of the slow reaction speed.

The use of F usarium solani or Streptomyces Zavendulae is also disadvantageous for the conversion of dehydroepiandrosterone to A -androstadiene-3,l7-dione because in addition to the desired A -androstadiene-3,17-dione also large amounts of testololactone and 1,2-dehydrotestololactone are formed.

It is accordingly a primary object of the present invention to provide for the production of A -steroids in high yields and with improved reaction speed.

It is another object of the present invention to provide a microbiological method of producing A -steroids which can start from various different starting compounds which could not be used as starting compounds prior to this method.

It is yet another object of the present invention to provide for the use of a new mutant of a microorganism which is particularly advantageous in the production of A -steroids.

A still further object of the present invention provides for the method of producing such microorganisms mutant for this purpose.

As yet another object the present invention provides for the production of new compounds which could not be produced prior to the method of the present invention.

Other objects and advantages of the present invention will be apparent from -a further reading of the specification and of the appended claims.

With the above and other objects in view, the present invention mainly comprises the discovery that a specially produced mutant of the Bacillus lentus has extremely good properties in the production of A -steroids starting from various different starting compounds.

It has been discovered that it is possible to produce from per se inactive strains of the family Bacillaceae, preferably of the genus Bacillus, specially of the species Bacillus lemus by choice artificially produced mutants of the species which exhibit a remarkably improved biochemical activity.

It has thus been discovered that by ultra violet irradiation of a strain of the species Bacillus lentils which with respect to 1,2-dehydrogenase activity has at most only slight activity, which is isolated from a compost, to obtain by systematic selection a mutant (MB 284) which not only has the desired activity of dehydrogenation in the 1,2-position in singular manner, but in addition is capable of converting a 3-hydroxy-A -group or a 3-acy1- oxy-A -group into the 3-keto-A -group. This obviously means a great extension of the groups of suitable starting materials for the production of A -3-ketosteroids.

The starting compounds for the method of the present invention may be designated as any steroid the A ring of which has the following structure:

wherein either Y or Z designates a carbon-carbon double 3 bond to the S-carbon atom, and wherein X is selected from the group consisting of By treatment of such starting compound with Bacillus lentus MB 284 in accordance with the method of the present invention the resulting compound will be the corresponding steroid wherein the A ring has the following structure:

A culture of Bacillus lentus MB 284 was deposited at the American Type Culture Collection in Washington, D.C., under No. 13805.

The surprising property of Bacillus lentus MB 284 does not correspond with any other known strain of the family Bacillaceae, and it also could not be predicted that by artificial mutation of a per se inactive strain that it would be possible to obtain a mutant with improved biochemical properties.

It has further been found that upon fermentation with the mutant MB 284 not only are surprisingly higher yields obtained, but also undesired conversion of the D-ring to a lactone ring does not occur and as a result the speed of the reaction is increased by about times to about 7 times.

Within the above mentioned broadened group of suitable starting materials are included perhydrocyclopentenophenanthrene compounds which are saturated in the 1,2-position which contain at least one hydrogen atom on each of the carbon atoms in 1 and 2 position, preferably however containing 2 hydrogen atoms on each of the 1- and Z-car-bon atoms, as well as compounds which contain on one of the two carbon atoms one hydrogen atom and on the other two hydrogen atoms, which compounds also contain an oxygen-function on the 3-carbon atom, preferably a keto group, a hydroxy group or an acyloxy group, most preferably a lower acyloxy group such as the acetyloxy group, in combination with a double bond between the 4 and 5 carbon atoms or between the 5 and 6 carbon atoms. It should be noted that these conditions do not exclude the possibility that at another portion of the steroid molecule there may be present other double bonds, and/or on the IO-carbon atom a methyl group, a free or functionally changed hydroxyl group or only a hydrogen atom, and that on still other carbon atoms such as the carbon atoms in the 6-, 9-, 11-, 16-, 17-, 20- or 21-position of the steroid framework there may be present the usual substituents such as keto groups, hydroxyl groups, methyl groups, epoxy groups, or halogens, particularly fluorine.

The further biochemical properties of the Bacillus lentus mutant MB 284 of the present invention are set forth in the following table in comparison to the species Bacillus lentus (according to Bergeys Manual of Determinative Bacteriology, 1957):

4 TABLE I Bacillus lentils Bacillus lentils M B 284 Milk agar ruled plate Milk Potato Starch ruled plate Acetylmethylcarbinol.

0.5% sodium citrate, 0.5% ammonium nitrate N 0 growth Slight growth Good growth, uniform turbidity, granular sediment.

Caseinis not hydrolyzed.

No growth Very slight growth.

Slight growth.

Good growth, uniform turbidity.

Casein not hydrolyzed. Unchanged. Slight growth. No hydrolysis. Is not formed.

N 0 growth.

Nitrate reduction Is not formed, no gas Is not formed, no

gas formation. gas formation. High gelatin1ayer No liquification N o liquiflcation. Gelatin plate do D Sodium nutrient 4% good, 5% no growth 211% good, 12%

212%. slight growth. Gas formation N one. None.

Ar. In the presence of 0 Xy. ammonium Meat extract plus Gl. salts as nitro- 0.5% ammonium- La. gen gel no acid 0 chloride. Ma formation So. occurs. Sc. Ar 0 Xy. 0 1% meat extract plus G1. 1% peptone. La. 0 Ma. 0 So. 0 Sc.

The following abbreviations are used in the table:

Ar. arnbinose. 'Xy. xylose. G1. glucose. La. lactose. Ma. mannose.

S0.:sorbosc.

Sc. sacclrarose. :acid formation. 0:no acid formation, =s1ight acid formation.

Examples GENERAL METHOD A 50 liter capacity stainless steel fermenter is charged with 30 liters of the nutrient solutions mentioned in the following table; the nutrient solution is sterilized by heating for one half hour at C., and after cooling inoculated with a bacteria suspension which is obtained by rinsing a bouillon agar surface of 64 cm. with 7 cc. of physiological saline solution.

After 2 days of culturing at 25 C. under stirring (220 revolutions per minute) and airing (1650 liters per hour) 1.8 liters of the resulting culture are removed under sterile conditions and transferred into a fermenter with 28.2 liters of the same medium. At the same time there is added a solution of 7.5 g. of the steroid mentioned in the table in 200 cc. of ethanol and fermented under the same conditions. The fermentation times depending upon the particular steroid are also set forth in the table, and it will be seen that these times vary.

The course of the fermentation is followed by removing samples which are extracted with methylisobutylketone. The extracts are analyzed by paper chromatography, preferably using a system of dioxane-i-toluene/propylene glycol and heptane/propylene glycol.

At the end of the fermentation time the culture broth is extracted three times, each time with 10 liters of methylisobutyl ketone. The purified extracts are concentrated in a circulating evaporator under vacuum and then under vacuum in a nitrogen atmosphere evaporated to dryness. The residue as subjected to chromatography on silica gel (10% addition of water). The utilized eluation agent TABLE 2-Cont1nued Analyt- Ferically Reaction Culture mentadeter- Eluation o Ex Substrate product; Medium timein tion mined agent olvent OLD solpfloll hours timein yield,

hours per cent 20--- lda-methyl-etlfia-methyl- 7 48 22 37 Groom"-.- Methanol. 217.5/219220...- m=

pregnene- 1,4-pregna- 17a-01-3,11, diene-17a- -trione ol-3,11,20- (mp. 211.5- trlone. 212.510. 23s=1 ,740 21 lfia-methylllfia-methyl- 7 4s 20 45 011013: Ethyl 176/179-1 1 m1= pregnen-21- l,4prcgnaethyl aceacetate/ ol-3,11,20- diene-21-oltate (4:1). hexane. trione+ 3,11,20- (m.p. 179- trione. 181 C 120) e237=15, 22.-- 16a-methyl-4- lfitz-methyl- 7 4a 22 82 Not chro- Ethyl 199-202 m= fl pregncne- 1,4-pregnamatcacetate. 17a-21-dio1- dime-17agraphed. 3,11,20-trl- 21-di0l-3,11, one. 20-trione.

1 20% starting material. 7 30% A4 compound.

The starting compound or substrate of Example 20, i.e. 16a-methyl-4-pregnene-17a-ol-3,l1,20-trione, is produced from the corresponding llfi-hydroxylated compound by oxidation with N-bromoacetamide in per se known manner.

The reaction products of Examples 19, 20 and 21 were acetylated in per se known manner to produce the following compounds:

160: methyl 1,4 pregnadiene 115,21 diol 3,20-

dione-Zl-acetate having a melting point of 204-205 C. (ethanol); e =15,020;

16a methyl 1,4 pregnadiene 17a ol 3,11 20- trione-l7a-acetate having a melting point of 225 .5-226 C. (chloroform/isopropyl ether); e =14,730;

16cc methyl 1,4 pregnadiene 21 o1 3,11 20- trione-Zl-acetate having a melting point of 2092l0 C. (ethanol); 62 q=14,820'.

The compounds of Examples 1, 2, 9, 13, 14, 15, 18, 19, 20, 21 and 22 possess corticoid activity, particularly an anti-inflammation action. The compound of Example 10 is anabolic. The compound of Example 17 is antiandrogenic, and the compounds of Examples 3, 4, 5, 6, 7, 8, 11, 12, 16 and 17 are suitable intermediate products for the synthesis of valuable steroids. These steroids can for example be produced by hydrogenating the 4,5-double bond, by acetylation of one or more hydroxyl groups, etc.

The fermentation temperature may in general vary between about 20 and 30 C., and is most preferably main tained at 25 C. The preferred concentration range is between 100 and 200 mg./l., and is most preferably about 250 mg. per liter.

Although the above set forth fermentations were all carried out with cultures, as indicated previously the reaction may be carried out by the use of enzymes which can be obtained in per se known manner by mechanically destroying the cells or by destroying them by means of ultrasonics and subsequently fractionally precipitating (for example by means of ammonium sulfate, protamine sulfate, etc.) or by fractional absorption (for example with calcium phosphate gel). This is preferably carried out at temperatures between 0 and 10 C.

Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can by applying current knowledge readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this invention and, therefore, such adaptations should and are intended to be comprehended within the meaning and range of equivalence of the following claims.

What is claimed as new and desired to be secured by Letters Patent is:

1. The method of producing 1,4-pregnadiene-11fl,17a, 2l-triol-3,20-dione, which comprises treating 4-pregnenel1B,17a-21-triol-3,20-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

2. The method of producing 1,4-pregnadiene-17a,21- diol-3,ll,20-trione, which comprises treating 4-pregnenel7a,21-diol-3,1:1,20-trione with a material selected from the group consisting of a Bacillus lcntus MB 284 culture and the enzymes isolated therefrom.

3. The method of producing 1,4-pregnadiene-17a,21- diol-3,20-dione, which comprises treating 4-pregnene- 17a,21-diol-3,20-dione with a material selected from the group consisting of a Bacillus lcntus MB 284 culture and the enzymes isolated therefrom.

4. The method of producing 1,4-pregnadiene-l4a,l7a, 2l-triol-3,20-dione, which comprises treating 4-pregnene- 14a,17ot,21-tfiOl-3,20di0ll8 with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

5. The method of producing 1,4-pregnadiene-12fi,17a, 2l-triol-3,20-dione, which comprises treating 4-pregnene- 12c,l7a,2l-triol-3,20-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

6. The method of producing 1,4-pregnadiene-6/8,17a, 21-triol-3,20-dione, which comprises treating 4-pregnene- 65,l7a,2l-triol-3,20-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

7. The method of producing 1,4-androstadiene-3,17- dione, which comprises treating 5-androstene-3fi-ol-17- one with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

8. The method of producing 4-androstadiene-3,17-dione, which comprises treating 4-androstene-3,l7-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

9. The method of producing 9a-fiuoro-1,4-pregnadiene- 11,8,17a,2l-triol-3,20-dione, which comprises treating 9afiuoro-4-pregnene-Ill 8,17a,21-triol-3,20-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

10. The method of producing 17u-methyl-1,4-androstadiene-3-one, which comprises treating 17a-methyl-4- androstene-17 3-ol-3-one with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

11. The method of producing 16a-methyl-1,4-pregnadiene-3,20-dione, which comprises treating 16a-methyl-4- pregnene-3,20-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

12. The method of producing 16,17a-oxydo-1,4-pregnadiene-21-ol-3,2 -dione, which comprises treating 16, 17a-oxyd0-5-pregnene-3,21-diol-20-one-21-acetate with a material selected from the group consisting of a Bacillus le ntus MB 284 culture and the enzymes isolated therefrom.

13. The method of producing IGa-methyLIA-pregnadiene-17a-21-diol-3,20 dione, which comprises treating 16a-methyl-4-pregnene-17a,21-diol-3,20-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

14. The method of producing l6a-methyl-l,4-pregnadiene-l'l,8,17a,21-triol-3,20-dione, which comprises treating 16a methyl 4-pregnene 11/3,17oc,21 triol 3,20- dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

15. The method of producing 1-6a-methy1-9afluoro- 1,4-pregnadiene-11,6,17a-21-triol-3,20-dione, which comprises treating 16a-methyl-9a-fluoro-4-pregnene-1118,17a, 21-triol-3,20-dione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

16. The method of producing 1,4-androstadiene-3, 17- dione, which comprises treating -androstene-3B-ol-17- one-3-acetate with a material selected from the group consisting of a Bacillus lelntus MB 284 culture and the enzymes isolated therefrom.

17. The method of producing A -dehydro-testololactone, which comprises treating testololactone with a material selected from the group consisting of a Bacillus len'tus MB 284 culture and the enzymes isolate-d therefrom.

18. The method of producing 16a-methyl-L4-pregnadiene-l1,8-17a-diol-3,20-dione, which comprises treating 16wmethy1-4-pregnene 113,170; diol-3,20-dione with a material selected trom the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

19. The method of producing 16amethyl-1,4-pregnadiene-l1/3,21-dio1-3,20-dione, which comprises treating 16a-methy1-4-pregnene-11fl,21-dio1-3,20-dione with a material selected from. the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

20. The method ot producing 16a-m1ethyl- 1,4-pregnadiene 17a cl 3,11,20-trione, which comprises treating 16a methyl 4 pregnenle 17a ol3,11,20-trione (M.P. 211.5-212.5 C., e =14,740) with a material selected firom the group consisting (of a Bacillus lemus MB 284 culture and the enzymes isolated therefrom.

21. The method of producing l6a-methy.l-l,4-pregnadiene-21-o1-3,11,20-tri0ne, which comprises treating 16amethyl-4-pregnene- 21-ol-3,11,20-trione (M.P. 179-181 C., E23'1:15,120) with a material selected iirom the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

22. The method of producing '16a-methyl-1,4-pregnadiene-17a-21-diol-3,11,20-trione, which comprises treattug 16a-methy1-4pregnene-17a-21 diol 3,11,20 trione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom.

23. The method of producing 16amethyl-1,4-pregnadiene-l1[3,21-diol-3,20-dione-21 acetate, which comprises treating 16a-methyl-4-pregnene 1113,21 diol 3,20-diode with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom, thereby forming as reaction product methyl-1,4-pregnadiene-11B,21 diol-3,20-di one; and acet- ,ylati ng said reaction product.

24. The method of producing 16a-nrethyl-1,4-pregnadien'e-'17aoi-3,11,20-trione-17a-acetate, which comprises treating 16a-methyl-4-pregnene 11oz ol 3,11,20 trione with a material selected from the group consisting of a Bacillus lentus MB 284 culture and the enzymes isolated therefrom, thereby forming as reaction product 160:- methyl-l,4pregna1diene-17a-ol-3,11,20-niche; and acetylatin-g said reaction product.

25. The method of producing 16a-methyl-L4-pregnadiene-21-o1-3,1 1,20 tn'lone 21 acetate, which comprises treating 16a-methyl-4-pregnene-2lol-3,11,20-t1ione with a material selected (from the group: consisting of a Bacillus lenzus MB 284 culture and the enzymes isolated therefirom, thereby forming as reaction product 16a-methyl- 1,4-pregnadiene-21-ol-3,11,20-trione; and acetylating said reaction product.

26. A method of producing a 1,4-diene-3-ketosteroid, which comprises treating with a material selected from the group consisting of a Bacillus len tus MB 284 culture and the enzymes isolated therefrom a compound selected from the group consisting of 4-pregnene-11fi,17a,21- tri!ol-3,20-dione, 4pregnene17a,21-diol-3,11,20-niche, 4- pregnene-17a,21-diol-3,20-dione, 4-pregnene-14a,17u,21- triol-3,20-di-on-e, 4-pregnene 125,17a,21-triol-3,20dione, 4 pregnene-6B,17a,21 triol 3,20-dime, S-androstene-Iiflol-17-one, 4-androstene-3,17-dione, 9a-fiuoro-4-pregnene 1lB,17oc,21 triol 3,20 dione, 17a-methyl-4-androstene- I7BoI-3-one, 16a-methyl-4 preguene-3,ZO-dione, 16a,17aoxido-S-pregnene 3,21 dioi20 one21-acetate, 16a-methyi-4-pregnene 17a,21-idio1-3,20-dione, 17a-methy1-4-pregnene-l1B,17a,21-triol-3,20 dione, 16u-methyl-9a-fluoro-4- pregnene-l1B,17a,21-tri0l-3,20-dione, -5-androstene-3fiol- 17one-3-acetate, testololactone, 16a-methyl-4-pregnene- 11B,17a-dio1-3,20'dione, 16a-methyl-4pregnene 115,21- diol 3,20 dione, I6a-methyI-4-pregnene-I7a-ol-S,I1,20- trione, 16a-methyl-4-pregnene- 21-ol-3,1 1,2O-trione, 16amethyI-4pregnene -17u-21-di01-3,11,20-1I10I16 and dehydro-testolollactone, A -pregnene 11p,17adio1- 3,20-(hone and Aflpregnene-l1fi,21-diol-3,204dione.

27. The method which comprises subjecting a Bacillus lentus strain which is substantially inactive with respect to 1,2-dehydrogena1ion or a 1,2-position saturated perhydrocyclopentenophenanthrene compound to ultra violet irradiation, thereby forming a mutant which has biochemical activity with respect to 1,2-dehydrogenaltion; and selecting and culturing such mutant.

References Cited in the file of this patent UNITED STATES PATENTS 2,666,016 Heehter et a1. Jan. 12, 1954 2,756,179 Fried et a1. July 24, 1956 2,844,513 Wettstein et all July 22, 1958 2,867,636 Lincoln et a1. Jan. 6, 1959 2,887,499 Carvajail May 19, 1959 2,958,631 Ohamney et a1 Nov. 1, 1960 OTHER REFERENCES Meystre et al.: Helv. Ohim. Acta, 39, 734-742 (1956). Ringold et al.: J. Org. Chem., 21, 239-40 (1956). 

1. THE METHOD OF PRODUCING 1,4-PREGNADIENE-11B,17A, 21-TRIOL-3,20-DIONE, WHICH COMPRISES TREATING 4-PREGNENE11N,17A-21-TRIOL-3,20-DIONE WITH A MATERIAL SELECTED FROM THE GROUP CONSISTING OF A BACILLUS LENTUS MB 284 CULTURE AND THE ENZYMES ISOLATED THEREFROM. 